In Vitro Transformation of Protopanaxadiol Saponins in Human Intestinal Flora and Its Effect on Intestinal Flora

Protopanaxadiol (PPD)-type ginsenosides are the main ginsenosides in ginseng (Panax ginseng C. A. Meyer) with potential therapeutic effects on diseases related to intestinal flora imbalance. This study aimed to investigate the in vitro metabolism of protopanaxadiol ginsenosides in human intestinal flora and their effect on the flora. Rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q-TOF MS) was utilised for the transformation of ginsenoside constituents for sample identification. Using 16S rDNA gene sequencing technique, the effect of PPD-type ginsenosides on gut microflora was analysed based on the indices of microflora diversity and gut microflora. The sample was transformed for 6 h, and the metabolites were ginsenoside Rb1, Rc, Rb2, Rb3, CO, Gyp-IX, Gyp-XVII, CMc-1, F2, Rg3, CK, Rh2, and PPD. The metabolites were CK, Rh2, and PPD when the samples were transformed for 60 h. The intestinal microflora were subjected to high-throughput sequencing using the Illumina MiSeq 2500 sequencing platform. In comparison with the faecal sample from the blank group, the protopanaxadiol saponin group significantly increased the relative abundance of Firmicutes and significantly decreased Bacteroidetes and Proteobacteria at the phylum level, whereas it significantly increased the relative abundance of Prevotella_9, Faecalibacterium, and Dialister and significantly decreased Escherichia-Shigella, Dorea, and Lachnoclostridium at the genus level. This study provides a basis for the determination of the pharmacodynamic material basis and pharmacodynamic targets of PPD-type ginsenosides based on the intestinal flora.


Introduction
Ginseng (Panax ginseng C. A. Meyer) is a precious oriental herb that has been used in traditional Chinese medicine for thousands of years, both as a disease-healing drug and a general tonic. P. ginseng is thought to be helpful in providing immunity against COVID-19 [1]. Many ginseng-based foods such as ginseng beverages, ginseng candy, ginseng tea, and ginseng honey tablets have been gradually developed worldwide [2]. According to the type of aglycones' structure, ginsenosides are divided into three types, including the oleanane, protopanaxadiol (PPD), and protopanaxatriol (PPT) types [3]. PPD-type ginsenosides ( Figure 1 and Table 1) account for 45%-60% of the total ginsenosides [4]. ey can inhibit oxidant stress, enhance immunity, lower blood sugar, resist tumour cells, and exhibit antiinflammatory properties [5,6].
After oral administration, most ginsenosides can interact with the intestinal flora in the intestines, thus transforming into secondary ginsenosides and being absorbed by the body [7]. PPT-type ginsenosides can be metabolised by the human intestinal flora, and the transformation products are mainly formed by the loss of sugar residues to form transformed products, while the secondary ginsenosides and aglycones are the material basis for the pharmacological effects of ginseng in vivo [8]. Moreover, the gut microbiome includes all the microbes living in the human gut, affecting human health and disease treatment. e balance of intestinal flora is closely related to the health and disease of the host. Adjusting the imbalance of intestinal flora, promoting probiotics, inhibiting pathogenic bacteria, correcting the imbalance of intestinal flora, and achieving a new balance are among the biological mechanisms of ginseng for the treatment of diseases [9,10]. Previous research by this group showed that the extract of ginseng and the seed of Zizyphus jujuba var. spinosa changes the structure and diversity of gut microbiota in rats with spleen deficiency syndrome and balances the metabolic process [11].
In this study, the intestinal flora of healthy individuals was examined in vitro. Rapid resolution liquid chromatography-quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF MS) detection technology was used to fit the original ginseng glycol-type saponin group in the human intestinal flora in metabolic pathways. en, we analysed the original-type ginseng diol saponin group after the structure of intestinal flora; this study aimed to reveal the original ginseng glycol-type saponin group in the law of metabolism in the body, the metabolic characteristics, and the effect of the PPD-type ginsenosides on the structure of intestinal flora.

Preparation of Isolated
e remaining residues were re-extracted thrice with n-butanol for each extraction. en, the ethyl acetate and n-butanol layers were mixed homogeneously. Solvent recovery was conducted by decompression and then diluted to the desired volume with methanol. e solutions were filtered through a 0.22-μm filter membrane before being injected for analysis.

Collection of Faecal Specimens.
GAM and an appropriate amount of normal human gut microbiota liquid were added to the blank group (K). In the experimental group (P), the gut microbiota solution of GAM, human gut microbiota liquid, and PPD-type ginsenosides were added. Both groups were collected and placed in an anaerobic incubator for aseptic operation in an anaerobic environment. e samples were then cultured for 24 h under anaerobic conditions at 37°C and 100 rpm. e sample was centrifuged at 11,000 rpm for 5 min. e supernatant was discarded, and the precipitate was placed in liquid nitrogen for 30 min and stored in a refrigerator at −80°C.

Preparation of Faecal DNA for
High-roughput Sequencing Analysis. DNA was extracted using an MN NucleoSpin 96 Soil DNA kit, according to the manufacturer's instructions. e sample was treated and split; impurities were removed, inhibitors; DNA binding was conducted; the silicon matrix membrane was cleaned and dried; DNA was eluted; and the tube was transferred. e DNA was stored at −80°C until further processing. For the 16S rDNA using primers 27FAGG: (TTTGA-TYNTGGCTCAG) and 1492R: (TASGGHT-ACCTTGTTASGACTT), PCR reactions were performed in triplicate with 50 μL of mixture containing 2.5 μL of VnF, 2.5 μL of Q5 high-fidelity DNA polymerase, 1.0 μL of high GC Enhancer, 25 μL of buffer, 10 μL of dNTP, and 40-60 ng of template DNA. Initial denaturation was conducted at 95°C for 5 min, 95°C for 30 s, and 50°C for 30 s, with a final extension at 72°C for 7 min, followed by 30 cycles. Finally, all PCR products were quantified using ImageJ software and pooled together.
High-throughput sequencing analysis of bacterial rRNA genes was performed on the purified and pooled samples using the Illumina HiSeq 2500 platform (PE250) at Biomarker Technologies Corporation, Beijing, China.

Bioinformatics and Statistical Analyses.
USearch software was used to cluster tags at a 97% similarity level to obtain OTU, and taxonomic annotations were made for OTU based on Silva and Unite databases. e representative OTU sequences were compared with the microbial reference database to obtain the corresponding species' taxonomic information.
en, the community composition of each sample was counted at the phylum and genus levels, and the community structure of the sample at the phylum and genus taxonomic levels was plotted using QIIME software and R language tools.

Component Analysis of PPD Ginsenosides in the Intestinal
Flora. RRLC-Q-TOF MS was performed to qualitatively analyse different ginsenosides during the biotransformation of PPD-type ginsenosides, and the obtained PPD-type ginsenosides and the precise molecular weight and retention time of the transformation products were obtained. Using tandem mass spectrometry (MS/MS), the characteristic fragment information of the parent ions was obtained through the rupture of the parent ions at the appropriate collision energy. Based on the above information and comparison with relevant literature, the identified components and their MS data are listed in Table 2. . In combination with the results of mass spectrum analysis [12], M2, M3, and M4 were eluted at 20.924, 21.629, and 22.473 min, respectively, and their molecular formula was C 53 H 90 O 22 . us, M2, M3, and M4 were deduced to be ginsenosides Rc, Rb2, and Rb3, respectively. M6 is ginsenoside CO, M7 is gypenoside Gyp-IX, M8 is gypenoside Gyp-XVII, M9 is ginsenoside CMc-1, M11 is ginsenoside F2, M11 is ginsenoside Rg3, M12 is Compound K, M13 is ginsenoside Rh2, and M14 is ginsenoside PPD.

Alpha Diversity Analysis.
Shannon and rarefaction analyses revealed that each sample was adequately sequenced and tended to be saturated, and the sequencing depth of the gut microbial environment was sufficiently captured in each sample and suitable for further analysis (Figure 4(a) and 4(b)).

Beta Diversity Analysis.
Beta diversity analysis compared the similarity of species diversity among different samples. Principal component analysis (PCA) can be used to classify multiple samples and further demonstrate species diversity differences among samples. e two ranking PCs, PC1 and PC2, described 97.91% and 1.36% of the total variability in the original observations, respectively. e PCA results showed that the structural composition of gut microbiota was different between groups K and P ( Figure 5). e aggregation of samples suggested similarities within each sample within each group and independence between the two groups. Figure 6(a) shows the differences in the species structure of intestinal flora between the two groups at the phylum level. e bacterial flora structure of the two groups was mainly composed of four dominant phyla: Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. is result is consistent with the research results of Valentina and Cecilia [13]. Approximately 30.51% of sequences (Figure 6(a)) in mice from the K group were classified as belonging to the phylum Firmicutes, whereas only 34.59% of sequences in mice from the P group were classified as belonging to this phylum. e relative abundance of Proteobacteria was 62.60% and 51.65% in mice from the P and K groups, respectively. e relative abundance of Bacteroidetes was 2.60% in mice from the P group and 16.64% in mice from the K group. Overall, the P group conspicuously changed the microbial composition in the model group with more Firmicutes and Proteobacteria but fewer Bacteroidetes. Wang et al. [14] found that after DSS administration, the proportion of Firmicutes was significantly decreased in the model group. In contrast, in the ginseng treatment group, the proportion of Firmicutes increased. is finding was consistent with our results. Ginseng polysaccharides have a prebiotic-like effect on OACS rats by simultaneously stimulating the growth of most important probiotics. Bacteroides spp. ginseng polysaccharides improved the intestinal metabolism and absorption of certain ginsenosides and reinstated the perturbed holistic gut microbiota [15]. is finding is consistent with our results. After ginsenoside administration, the abundance of Bacteroidetes was significantly reduced. Hence, ginsenoside also has a regulatory effect on the intestinal flora of normal people and can prevent diarrhoea caused by the imbalance of bacterial flora.

Key Phylotypes of Gut Microbiota.
At the genus level ( Figure 6(b)), 11 bacterial genera were isolated from the three groups at the genus level. Bacteria with high relative abundance include Escherichia-Shigella, Sutterella, Streptococcus, Prevotella_9, Lachnoclostridium, and Megasphaera. Compared with group K, the relative abundance of Escherichia-Shigella in group P was increased, whereas that of Dorea, Prevotella_9, and Megasphaera decreased. In the K group, approximately 44.68% of the sequences were classified as Escherichia-Shigella, and Evidence-Based Complementary and Alternative Medicine 5 approximately 12.71% of the sequences were classified as Preprevella_9. Escherichia-Shigella had a high correlation with digestive tract symptoms. Hence, it also had a great influence on the "enteric-brain" axis. e treatment of mice with the probiotic bacterium Lactobacillus rhamnosus JB-1 reduces stress-and depression-like behaviour in a vagus nerve-dependent manner [16]. Administration of the original ginsenosides may ameliorate depression. Prevotella is the dominant bacteria in the intestinal tract, and Huws et al. [17] found that Prevotella is an inflammation-related bacterium that can cause diarrhoea. erefore, the administration of proginsenediol ginsenosides can prevent and treat diarrhoea. Song et al. [18] reported the effect of ginseng on the intestinal flora of middle-aged obese Korean women, in which the relative abundance of Dorea was high in obese people but decreased after ginseng was administered, indicating that Dorea is related to human obesity. Ginseng has significant effects on the treatment of obesity, and current research results can provide a basis for clinical treatment.
Linear discriminant analysis effect size (LEfSe) can determine the colony species with statistical differences among different groups. e histogram of LDA value distribution and the evolutionary branch diagram of LEfSe analysis showed that the LDA scores of the K and P groups were significantly higher than the present value of 4 (Figure 7(a) and 7(b)). A total of 31 species differed between the two groups. In the P group, o_Lactobacillales, c_Bacilli, g_Streptococcus, s_Streptococcus_salivarius, f_Streptococcaceae, f_Enterobacteriaceae, g_Escherichia_Shigella, o_Enterobacteriales, c_Gammaproteobacteria, and p_Proteobacteria were relatively abundant, and statistically significant differences were observed (p < 0.05). In the P group, g_Lach-nospiraceae_UCG_004, s_Eubacterium_hallii, s_Dakarella_massiliensis, g_Sutterella, f_Burkholderiaceae,  Evidence-Based Complementary and Alternative Medicine o_Bacteroidales, o_Betaproteobacteriales, f_Veillonellaceae, g_Megasphaera, o_Selenomonadales, c_Negasphaera, c_Bacteroidia, f_Prevotellaceae, o_Bacteroidales, g_Pre-votella_9, p_Firmicutes, o_Clostridiales, c_Clostridia, and p_Bacteroidetes were relatively abundant, and statistically significant differences were observed (p < 0.05). e relative abundances of p_Firmicutes, o_Clostridiales, and c_Clostridia were statistically different, and they all belonged to Firmicutes. e relative abundance of p_Bacteroidetes was statistically different and belonged to Bacteroidetes. e relative abundance of Firmicutes and Bacteroidetes in the gut microbiota of the PPD-type ginsenoside group was significantly different, which was consistent with the analysis of microbial diversity.
3.6. Functional Analysis of Gut Microbiota. KEGG pathway analysis is usually used to analyse the differences and changes in functional genes in the metabolic pathways between samples and groups. As shown in Figure 8,   Evidence-Based Complementary and Alternative Medicine significant differences were observed in seven metabolic categories of the intestinal flora in group P compared with group K. Among them, environmental information processing, cellular processes, and human diseases differed. In conclusion, the intestinal flora of the original ginsenoside group may be involved in this process. However, the correlation between the human intestinal flora and the microbes that changed in the intestinal flora after ginsenoside intervention and the KEGG metabolic pathway remains to be further studied.

Conclusions
RRLC-Q-TOF MS detection technology was used for the transformation of saponin constituents during sample identification. e human intestinal flora has a metabolic effect on the original ginsenoside; under the action of the intestinal flora in the human gut, the ginsenoside is mainly carried out via deglycosylation, and the ginsenoside is gradually metabolised into secondary aglycones and absorbed into the blood. During biotransformation, the metabolic form and time of the original ginsenosides were relatively stable. PPD-type ginsenosides and their metabolites were present when the sample was transformed to 6. When the sample was transformed for 60 h, most of it existed in the form of Compound K and ginsenoside Rh2, and a few were in the form of ginsenoside PPD. e structure of intestinal flora is complex, and the composition of intestinal flora varies from individual to individual. Bacteria are the main component of intestinal microorganisms, most of which belong to Firmicutes, Bacteroidetes, and Proteobacteria, among which Firmicutes is the dominant phylum. At the phylum level, the P group conspicuously changed the microbial composition in the model group with more Firmicutes and Proteobacteria but fewer Bacteroidetes. At the genus level, compared with group K, the relative abundance of Escherichia-Shigella in group P increased, whereas that of Dorea, Prevotella_ 9, and Megasphaera decreased. e proginsenediol saponin group increased the species composition of beneficial bacteria and decreased the relative abundance of harmful bacteria in the intestinal flora at the phylum and genus levels, and the improvement of the intestinal flora may play an important role in disease treatment and health care functions.
Data Availability e data that support the findings of this study are available from the corresponding author, H. Y., upon reasonable request.

Conflicts of Interest
e authors declare no conflicts of interest.